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ARTICLE IN PRESSTH-10336; No. of Pages 9

Atherosclerosis xxx (2008) xxx–xxx

Diphenyl diselenide, a simple glutathione peroxidase mimetic,inhibits human LDL oxidation in vitro

Andreza Fabro de Bem a,∗, Marcelo Farina a, Rafael de Lima Portella b,Cristina Wayne Nogueira b, Teresa C.P. Dinis c,Joao A.N. Laranjinha c, Leonor M. Almeida c,

Joao Batista Teixeira Rocha b

a Departamento de Bioquımica, Centro de Ciencias Biologicas, Universidade Federal de Santa Catarina, Florianopolis, SC 88040900, Brazilb Departamento de Quımica, Centro de Ciencias Naturais e Exatas, Universidade Federal de Santa Maria, Santa Maria, RS 97105900, Brazil

c Laboratorio de Bioquımica, Faculdade de Farmacia, Universidade de Coimbra, Coimbra 3000, Portugal

Received 29 August 2007; received in revised form 27 February 2008; accepted 29 February 2008

bstract

Oxidative modification of low-density lipoprotein (LDL) represents an important factor in atherogenesis. In the present study, we havenvestigated the antioxidant capability of diphenyl diselenide (PhSe)2, a simple organoseleno compound, against copper (Cu2+) and peroxyladical-induced human LDL oxidation in vitro. In initial studies using human serum, (PhSe)2 caused a dose-dependent inhibition of Cu2+-nduced lipid peroxidation, which was correlated to thiol consumption. (PhSe)2 increased lipid peroxidation lag phase and decreased lipideroxidation rate in isolated human LDL, evaluated by measuring both conjugated diene (CD) and thiobarbituric acid reactive substancesTBARS) levels. Consistent with these observations, (PhSe)2 showed a marked inhibitory effect on 2,2-azobis(2-amidinopropane dihydrochlo-ide) (AAPH)-induced oxidation of LDL or parinaric acid (PnA) incorporated into LDL. (PhSe)2 also displayed a dose-dependent protectiveffect against Cu2+-induced lipid peroxidation in rat aortic slices. Interestingly, besides the antioxidant effects of (PhSe)2 toward the lipidoieties of LDL, which was related to its thiol-peroxidase activity, protein moieties from human isolated LDL were also protected againstu2+-induced oxidation. The results presented herein are the first to show that (i) (PhSe)2 inhibits lipid peroxidation in human isolated LDL

n vitro, (ii) this phenomenon is related to its thiol-peroxidase activity, and (iii) this chalcogen also prevents the oxidation of protein moietiesf human LDL. Taken together, such data render (PhSe)2 a promising molecule for pharmacological studies with respect to the atherogenicrocess.

2008 Elsevier Ireland Ltd. All rights reserved.

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eywords: Diphenyl diselenide; Selenium; Ebselen; Glutathione peroxidas

. Introduction

Atherosclerosis remains the most common cause of deathn industrialized countries. This affects the vascular wall andeads to coronary artery diseases and cerebrovascular acci-ents (stroke) [1]. There is increasing evidence that oxidative

Please cite this article in press as: Bem AF, et al., Diphenyl diselenideoxidation in vitro, Atherosclerosis (2008), doi:10.1016/j.atherosclerosis.

odifications of low-density lipoprotein (LDL) play a pivotalole in the development of atherosclerosis. Moreover, ele-ated levels of oxidized LDL have been positively correlated

∗ Corresponding author. Tel.: +55 48 37219589; fax: +55 48 37219672.E-mail address: andrezadebem@ccb.ufsc.br (A.F. Bem).

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021-9150/$ – see front matter © 2008 Elsevier Ireland Ltd. All rights reserved.oi:10.1016/j.atherosclerosis.2008.02.030

oxidation; Atherosclerosis

o the severity of acute coronary events and have been con-idered a biochemical marker for coronary heart disease [2].

Although the molecular bases related to the triggering pro-ess of LDL oxidation remain unclear, the major mechanismsurrently explored are metal ions dyshomeostasis, changes inipoxygenase- and myeloperoxidase-related pathways, reac-ive oxygen and nitrogen species generation, and alterationsn the thiol status [3].

, a simple glutathione peroxidase mimetic, inhibits human LDL2008.02.030

Since LDL oxidation plays a key role in the pathogenesisf atherosclerosis, antioxidants that can inhibit this oxidativerocess might be useful in preventing atherosclerosis-relatedathological conditions, such as coronary artery diseases and

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ARTICLEA.F. Bem et al. / Athero

troke [4]. In this regard, it has been evidenced that antiox-dant capability of LDL can be easily increased by dietaryntioxidant supplementation. In fact, many endogenous andxogenous compounds have been reported to display benefi-ial effects against LDL oxidation [5].

Selenium (Se) is an essential nutrient associated with theunction of major metabolic pathways in the cell, where it isncorporated as selenocysteine at the active site of a wideange of proteins [6]. Selenium intake is inversely corre-ated to the incidence of atherosclerosis and coronary heartisease [7]. In endothelial cells, selenium can protect fromxidative damage by altering the expression of selenoproteinshich have antioxidant function, including cytoplasmic glu-

athione peroxidase (cyGPX), phospholipid hydroperoxidelutathione peroxidase (PHGPx) and thioredoxin reductaseTR) [8].

The interest in organoselenium chemistry and biochem-stry has increased in the last three decades mainly dueo the fact that several organoselenium compounds pos-ess antioxidant activity [9], which is interplayed with itslutathione-peroxidase-like activity [10,11]. The first exam-le of such a compound was ebselen [12,13], and thisompound has been extensively studied in reason of itsntioxidants and anti-inflammatory properties [14,15]. Con-idering the pharmacological properties of ebselen, we haveeen studying the pharmacological properties of anotherrganoselenium compound, diphenyl diselenide (PhSe)2,hich also exhibits thiol-peroxidase-like activity which is

bout two times that of ebselen [10,16].This organoselenium caused minimal toxicity when

dministrated acutely to mice and rats [17] and after a longerm exposure to rabbits [18,19] and has been reported toresent lower toxicity than ebselen [11]. Recently, studiesrom our laboratory have demonstrated that (PhSe)2 causedsignificant reduction in blood glucose and glycated protein

evels in diabetic rats [20]. Moreover, the in vitro and in vivontioxidant potential of (PhSe)2 was already demonstrated ineveral tissues and models, including brain, liver and platelets11,21].

Given the earlier observations, the present study was car-ied out to evaluate the potential beneficial effects of (PhSe)2n protecting in vitro oxidation using different methodologi-al approaches: human serum, human isolated LDL, and ratortic slices (rich in endothelial cells), which are the mainlements involved in the early atherosclerosis developmentn vivo. The thiol-peroxidase capability of (PhSe)2 was alsovaluated in an attempt to delve into molecular mechanismselated to the aforementioned antioxidant effects.

. Materials and methods

Please cite this article in press as: Bem AF, et al., Diphenyl diselenideoxidation in vitro, Atherosclerosis (2008), doi:10.1016/j.atherosclerosis.

.1. Materials

Diphenyl diselenide (PhSe)2 was synthesized accordingo a method previously described [22]. Analyses of the 1H

atce

PRESSis xxx (2008) xxx–xxx

MR and 13C NMR spectra showed that the obtained com-ound had analytical and spectroscopic data in full agreementith its assigned structure. The chemical purity of (PhSe)2

99.9%) was determined by GC/HPLC. (PhSe)2 was solubi-ized in ethanol PA and a 10-mM stock solution was stored at◦C for less than 2 weeks. Immediately before use, (PhSe)2as diluted in ethanol PA at the required concentrations for

he different assays. All other chemicals were of analyticalrade and obtained from standard commercial suppliers.

.2. Serum oxidation

The study was approved by our Ethic Committee atniversidade Federal de Santa Maria. Blood samples were

ollected from healthy and normolipidemic volunteers after12-h overnight fasting period. Samples were left to clot

n the dark at room temperature for 30 min and then cen-rifuged at 1500 × g for 15 min. The serum was removed andmmediately used in the oxidation assays and oxidations wereerformed in serum rather than in plasma to avoid poten-ial interferences of substances such as EDTA, heparin, oritrate. Briefly, serum samples were diluted 1:4 in 10 mMotassium phosphate buffer, pH 7.4 and incubated at 37 ◦Cith CuSO4 (100 �M) and/or (PhSe)2 (0–200 �M). The totalolume was 9 mL. At different time points, aliquots (200 �L)ere removed for evaluating thiobarbituric acid reactive sub-

tances (TBARS) levels and consumption of the total thiolroups (–SH). Serum TBARS levels and thiol groups wereetermined based on Ohkawa et al. [23] and Ellman [24],espectively.

.3. Thiol-peroxidase activity of (PhSe)2 andpectroscopy studies

The thiol-peroxidase activity of (PhSe)2 was measuredccording to a method previously described by Wilson et al.10]. (PhSe)2 (50 �M) was incubated at 37 ◦C in a mediumontaining 50 mM potassium phosphate buffer, pH 7.0, 1 mMthylene diamine tetraacetic acid (EDTA), 1 mM reducedlutathione (GSH), 1 U of GR and 0.25 mM NADPH (finalolume of reaction = 1 mL). The reaction was initiated byddition of 0.5 �mol of hydrogen peroxide. The activity wasollowed by the decrease of NADPH absorption at 340 nm.ppropriate controls were carried out without (PhSe)2 andere subtracted.In order to evaluate the potential chemical interaction

etween (PhSe)2, GSH and H2O2, (PhSe)2 (20 �M) wasncubated with GSH (200 �M) in 10 mM potassium phos-hate buffer (pH 7.4). The reaction was performed at 37 ◦Cn a quartz cuvette and monitored spectrophotometrically250–400 nm) using a PerkinElmer Lambda 6 spectropho-ometer. In additional experiments, H O (200 �M) was

, a simple glutathione peroxidase mimetic, inhibits human LDL2008.02.030

dded 1 min after the reaction of (PhSe)2 with GSH. The reac-ion mixture had 2 mL. Temperature was maintained using airculating water bath. Readings were done against a refer-nce cuvette containing phosphate buffer.

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.4. LDL isolation

LDL was isolated from fresh human plasma by discon-inuous density-gradient ultracentrifugation as described byilva et al. [25], with slight modifications. Plasma of non-asted healthy normolipidemic donors collected with EDTA1 mg/mL) was pooled and sucrose (final concentration,.5%) was added to prevent LDL aggregation. Five millilitersf EDTA–plasma adjusted to a density of 1.22 g/mL witholid KBr (0.326 g/mL) was layered on the bottom of a cen-rifuge tube. Then 5 mL EDTA-containing sodium chlorideolution (density 1.006 g/mL) was overlaid on top of thelasma. Ultracentrifugation was run at 65,000 rpm for 2 ht 4 ◦C, in a Hitachi ultracentrifuge. LDL particles were col-ected by aspiration of the yellow band, which is located inhe middle of sodium chloride solution just above the plasma

ain fractions. Then, 4 mL of LDL particles was dialyzedxhaustively overnight at 4 ◦C against 6 L of 10 mM phos-hate buffer to remove the excess salt and the majority ofhe EDTA. Protein concentration in LDL solution was deter-

ined by Lowry et al. [26]. The purity of LDL preparationas verified by agarose gel electrophoresis. Isolated LDLas stored at −20 ◦C not longer than 2 weeks.

.5. LDL oxidation

Incubation of LDL with copper ions initiates lipid per-xidation and causes extensive oxidation of the LDL lipids.u2+-mediated oxidation is frequently used to assess the sus-eptibility of LDL to oxidation which is regarded as a possibleisk factor for atherosclerosis [27].

LDL oxidation was monitored by following the CD for-ation and the TBARS production. LDL samples (50 �g

rotein/mL) were pre-incubated at 37 ◦C in a medium con-aining 10 mM potassium phosphate buffer, pH 7.4 andifferent concentrations of (PhSe)2 (0–40 �M). After 10 min,uSO4 (1.6, 5 or 10 �M) was added to the reaction mediumnd aliquots were removed at different time points forvaluating CD and TBARS production. In another set ofxperiments, (PhSe)2 (40 �M) was added at different timeoints (0, 45 or 90 min) in an attempt to evaluate whetherPhSe)2 could inhibit Cu2+-induced LDL oxidation once therocess was started.

Potential antioxidant effects of (PhSe)2 against LDLxidation were also evaluated after the addition of anzo initiator, 2,2-azobis(2-amidinopropane dihydrochloride)AAPH) (1 mM), whose slow decomposition generates per-xyl radical at a constant rate in aqueous medium and induceshe chain oxidation of human LDL by a free radical mediated

echanism.In the studies of CD formation, the temporal change in

bsorbance at 234 nm (which refers to the CD concentration)

Please cite this article in press as: Bem AF, et al., Diphenyl diselenideoxidation in vitro, Atherosclerosis (2008), doi:10.1016/j.atherosclerosis.

s divided into three phases: a lag phase, a propagation phase,nd a terminal phase. The lag phase was defined as the inter-ept of the tangent of the slope of the absorbance curve inropagation phase with the time axis, and was expressed in

bKav

PRESSis xxx (2008) xxx–xxx 3

in. The rate of propagation was obtained from the slopef the absorbance curve during the propagation phase, andhe terminal phase corresponds to the extensive oxidation ofipids of LDL. Absorbance at 234 nm was normalized withespect to the absorbance at the beginning of the oxidationrocess.

.6. Measurement of parinaric acid (PnA) fluorescence

PnA is a polyunsaturated fatty acid that has been used suc-essfully as a fluorescent probe to monitor the initial stagesf lipid peroxidation by peroxyl radicals from AAPH [28].is-PnA (9,11,13,15-octadecatetraenoic acid) fluorescenceas monitored in a PerkinElmer LS 50 spectrofluorometerrovided with a thermostated cuvette containing a magnetictirring device. The excitation and emission wavelengthsere 324 and 413 nm, respectively (slit widths: 3.5 nm).Preliminary studies were performed in order to deter-

ine the concentration of LDL and PnA that insure totallyncorporation of the probe into LDL and a linear fluorescentesponse with PnA concentration. The assays were performedt 37 ◦C in 2 mL of potassium phosphate buffer (110 mMaCl, 20 mM phosphate, pH 7.4) containing 45 �g of LDLrotein and an aliquot of ethanolic solution of PnA (1.5 �Mnal concentration). The incorporation of the probe was car-ied out by gentle stirring for 1 min. Increasing concentrationsf (PhSe)2 were added to the mixture and the oxidationeaction was initiated by addition of AAPH (10 mM finaloncentration). The capacity of (PhSe)2 to protect LDL fromxidation was determined by the inhibition of probe fluores-ence decay [28].

.7. Measurement of LDL-Trp fluorescence

The time course of tryptophan (Trp) fluorescence emis-ion intensity is used to monitor Cu2+-induced apolipoproteinDL oxidation. The fluorescence spectra of native LDL dis-lay a single band centered at approximately 332 nm, which isssigned to the Trp residues in apo B. Loss of Trp fluorescences a marker for oxidations at the protein core of LDL [29].u2+-induced LDL oxidation was performed in a similarlyay to item described in Section 2.5, excepting that 3.3 �MuSO4 and different (PhSe)2 concentrations (0–30 �M) weresed. Trp fluorescence was measured at different time points0–360 min) using a Shimatzo spectrofluorometer (excitationt 282 nm and emission at 331 nm).

.8. Aortic slices oxidation

Adult Wistar rats were decapitated, thoracic aorta wereuickly dissected and then removed, rinsed and submergedn a petri dish filled with ice-cold isosmotic phosphate

, a simple glutathione peroxidase mimetic, inhibits human LDL2008.02.030

uffer (NaCl 124 mM, Na2HPO4 10 mM, NaH2PO4 5 mM,H2PO4 5 mM, glucose 10 mM, pH 7.4) and cleaned of

dherent fat and connective tissue in an ice-bath. Trans-erse propsections (400 �m) were prepared using a McIlwain

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4 sclerosis xxx (2008) xxx–xxx

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Fig. 1. (A) Effects of (PhSe)2 on Cu2+-induced lipid peroxidation and thiolconsumption in human serum. At different time points (indicated in theabscissa axis) TBARS (solids line) and SH (broken lines) content wereanalyzed (see Section 2). TBARS levels are expressed as nmol of mal-ondialdehyde (MDA). Total thiol (–SH) content is expressed as percentof control, whose basal concentration was 437 ± 25 nmol/mL. Results arerepresented as mean ± standard error of mean (S.E.M.) from at least threeindependent experiments. (B) The comparative spectra of (PhSe)2 and theproducts of its interaction with GSH and/or H2O2. (A) (PhSe)2 (20 �M);(B) GSH (200 �M); (C) (PhSe)2 (20 �M) + GSH (200 �M) + 1 min at 37 ◦C;(D) (PhSe)2 (20 �M) + GSH (200 �M) + 1 min + H2O2 (200 �M) + 1 min at3ag

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ARTICLEA.F. Bem et al. / Athero

issue chopper. Aortic slices (10 slices) were incubated insosmotic phosphate buffer with 20 or 40 �M of (PhSe)2nd/or 10 �M CuSO4 for 2 h. After incubations, aortalices were washed with saline and homogenized in 400 �Lf acetic acid buffer (pH 3.5). Lipid oxidation in aortaomogenates was measured by determining TBARS, asescribed by Ohkawa et al. [23]. Experimental proceduresnvolving animals were approved by the local Animal Careommittee.

.9. Statistical analysis

Values are expressed as mean ± S.E.M. One-way analysisf variance (ANOVA) was used for multiple comparisons,ollowed by the Duncan’s multiple range test when the F-alue was significant. Only significant F-values are givenn the text. Linear regression analysis was also used to testose-dependent effects. All analyses were performed usinghe Statistical Package for the Social Sciences (SPSS) soft-are in a PC-compatible computer. A value of p < 0.05 was

onsidered to be significant.

. Results

.1. Effects of (PhSe)2 on serum oxidation

The protective effects of (PhSe)2 against Cu2+-inducedBARS generation and thiol consumption in blood serumre depicted in Fig. 1A. Cu2+ (100 �M) caused a time-ependent increase of serum TBARS levels (Fig. 1A, right,axis) and a time-dependent decrease of total thiol groups

Fig. 1A, left, Y axis). Interestingly, even though (PhSe)2isplayed a concentration-dependent inhibitory effect towardu2+-induced TBARS generation (F(4,10) = 21.18; p < 0.001;= −0.877), there was a stimulation of thiol groups con-

umption by (PhSe)2.

.2. Spectroscopy studies

In order to delve into molecular mechanisms involvedith the beneficial role of (PhSe)2 against LDL lipid per-xidation, in vitro experiments of light/UV spectroscopyoncerning the chemical interaction between (PhSe)2, GSHnd H2O2 were investigated spectrophotometrically. Theharacteristic spectra of 20 �M (PhSe)2 (line A, Fig. 1B)nd of 200 �M GSH (line B, Fig. 1B) was changed afterheir reaction (line C, Fig. 1B), probably due to the forma-ion of phenyl selenol intermediate (PhSe−). Interestingly,fter the addition of H2O2 (200 �M), the strong and broadbsorption peak at 270 nm of such intermediate was abol-shed, indicating its chemical interaction with H2O2 (Fig. 1B,

Please cite this article in press as: Bem AF, et al., Diphenyl diselenideoxidation in vitro, Atherosclerosis (2008), doi:10.1016/j.atherosclerosis.

pectrogram D). Consistent with this observation, (PhSe)2isplayed a concentration-dependent glutathione peroxidase-ike activity (Fig. 1B, inset), which was indirectly measuredy NADPH consumption in the presence of GSH, purified

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7 ◦C. Spectra of 20 �M (PhSe)2 or 200 �M GSH were not modified byddition of 200 �M H2O2. For details, see Section 2. The inset graph showslutathione peroxidase activity of (PhSe)2.

SH reductase and hydrogen peroxide. The peroxidase-likectivity of (PhSe)2 was approximately twice when comparedo that of ebselen (data not show), these results are in agree-

ent with the previous study of Wilson et al. [10], wherehey showed that (PhSe)2 is about 1.6 times more effectives GPx-mimetic than ebselen. Moreover, (PhSe)2 catalyti-ally increased GSH oxidation in the presence of H2O2 in

, a simple glutathione peroxidase mimetic, inhibits human LDL2008.02.030

concentration-dependent manner (data not show). Takenogether, these data indicate that the chemical interactionetween (PhSe)2 and GSH produces an intermediate that isapable of interacting with peroxides. This process allows for

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Fig. 2. (A) Effects of (PhSe)2 on AAPH-induced conjugated dienes for-mation in human LDL. At different time points, conjugated dienes wereanalyzed. LDL samples (50 �g protein/mL) were incubated at 37 ◦C with1.0 mM AAPH in the absence (control) or presence (3–50 �M) of (PhSe)2

(broken lines represent control conditions without Cu2+ addition). Con-jugated dienes are expressed as absorbance at 234 nm. Inset shows thesignificant correlation between oxidation rate and (PhSe)2 concentrations(F(7,16) = 83.21; p < 0.001; β = −0.944). (B) Effect of (PhSe)2 on AAPH-induced oxidation of PnA incorporated into LDL. LDL samples (45 �gprotein) were incubated at 37 ◦C with 1.5 �M PnA for 1 min under gen-tle stirring. AAPH (1.0 mM) and (PhSe)2 (0, 2, 3, or 4 �M; 3 min afterAAPH addition) were added to the reaction medium. Inset shows a typicalcontrol assay of PnA oxidation, incorporated into LDL, initiated by AAPH.The fluorescence intensity was recorded up to 30 min and shows the initiallight scattering and the fluorescent signal decay following AAPH addition.Rw

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ARTICLEA.F. Bem et al. / Athero

he detoxification of peroxides at the expenses of sulfhydrylroups from GSH.

.3. Effects of (PhSe)2 on the LDL oxidation

.3.1. (PhSe)2 effects on AAPH-mediated lipid LDLxidation

The effects of (PhSe)2 on AAPH-mediated lipidDL oxidation is depicted in Fig. 2A. AAPH caused

significant increase of CD formation within timeFig. 2A). (PhSe)2 (3–50 �M) showed a marked inhibitoryffect on AAPH-induced lipid LDL oxidation in aoncentration-dependent manner. In fact, a significant neg-tive correlation (F(7,16) = 83.21; p < 0.001; β = −0.944) wasbserved for (PhSe)2 concentrations and CD levels (insetig. 2A).

.3.2. (PhSe)2 effects on PnA fluorescenceFig. 2B shows that low concentrations of (PhSe)2

2–4 �M) caused a concentration-dependent inhibitionF(3,8) = 684.27; p < 0.001; β = 0.215) of the fluorescencentensity decay of PnA in the presence of AAPH when com-ared to the control condition (absence of (PhSe)2). The insetf Fig. 2B indicates a typical control assay where the decreasef the fluorescence of PnA following the addition of AAPHeflects its oxidative degradation.

.3.3. (PhSe)2 effects on Cu2+-induced LDL oxidationThe effects of (PhSe)2 on Cu2+-induced lipid oxidation

n isolated LDL are depicted in Fig. 3. (PhSe)2 inhib-ted Cu2+-induced generation of both CDs (Fig. 3, left)nd TBARS (Fig. 3, right) in a concentration-dependentanner. Cu2+ displayed concentration-dependent oxida-

ive effects toward lipids, evaluated by measuring CDsFig. 3). LDL oxidation showed an expected oxidationattern of an initial lag phase followed by a propa-ation phase and a decomposition phase (Fig. 3, left),s originally described by Esterbauer et al. [4]. Interest-ngly, (PhSe)2 caused concentration-dependent increasesn lag phase and decreases in the oxidation rate, evi-enced by changes in the propagation phase slopeCu2+ 1.6 �M: F(3,8) = 104.05; p < 0.001; β = −0.910; Cu2+

�M: F(3,8) = 650.72; p < 0.001; β = −0.956; Cu2+ 10 �M:(3,8) = 41.46; p < 0.001; β = −0.932).

In another set of experiments, (PhSe)2 (40 �M) was addedt different time points (0, 45 or 90 min) in an attempt tovaluate whether (PhSe)2 could inhibit Cu2+-induced lipidDL oxidation once the process was started. Fig. 4A shows aignificant inhibitory effect of (PhSe)2 toward Cu2+-inducedipid LDL oxidation when present at the beginning of the

Please cite this article in press as: Bem AF, et al., Diphenyl diselenideoxidation in vitro, Atherosclerosis (2008), doi:10.1016/j.atherosclerosis.

xidation process. Interestingly, (PhSe)2 inhibited Cu2+-nduced lipid LDL oxidation when added to the reaction

edium at 45 and 90 min after the oxidation process wastarted.

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esults are derived from a single representative experiment. Experimentsere repeated at least three times, showing similar results.

.4. Effects of (PhSe)2 on the LDL-Trp fluorescencemission

Fig. 4B shows that protein moieties of LDL are oxi-

, a simple glutathione peroxidase mimetic, inhibits human LDL2008.02.030

ized within time in the presence of CuSO4 (3.3 �M). Thishenomenon was prevented by (PhSe)2 in a concentration-ependent manner (F(3,8) = 104.05; p < 0.001; β = 0.954). Its noteworthy that Cu2+-induced apolipoprotein LDL oxida-

Please cite this article in press as: Bem AF, et al., Diphenyl diselenide, a simple glutathione peroxidase mimetic, inhibits human LDLoxidation in vitro, Atherosclerosis (2008), doi:10.1016/j.atherosclerosis.2008.02.030

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Fig. 3. Effects of (PhSe)2 on Cu2+-induced lipid peroxidation in human LDL. At different time points (indicated in the abscissa axis), conjugated dienes (left)and TBARS (right) were analyzed. LDL samples (50 �g protein/mL) were incubated in the presence of 1.6 �M (A), 5.0 �M (B) and 10 �M (C) CuSO4 and inthe absence (control) or presence (1–40 �M) of (PhSe)2 (broken lines represent control conditions without Cu2+ addition). TBARS levels are expressed as nmolof malondialdehyde (MDA). Conjugated dienes are expressed as absorbance at 234 nm. Results are represented as mean ± standard error of mean (S.E.M.)from at least three independent experiments.

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Fig. 4. (A) Effects of (PhSe)2 on Cu2+-induced conjugated dienes formationin previously oxidized human LDL. LDL samples (50 �g protein/mL) wereincubated at 37 ◦C in the presence of 1.6 �M CuSO4 and in the absence (con-trol) or presence (40 �M) of (PhSe)2, which was added at 0, 45 or 90 minafter CuSO4 addition. At different time points (indicated in the abscissa axis),conjugated dienes were analyzed. Results are expressed as absorbance at234 nm. Results are derived from a single representative experiment. Exper-iments were repeated at least three times, showing similar results. (B) Effectsof (PhSe)2 on Cu2+-induced loss of tryptophan fluorescence in human LDL.LDL samples (50 �g protein/mL) were incubated at 37 ◦C in the presence of3.3 �M CuSO4 and different (PhSe)2 concentrations (0–30 �M). Tryptophanfluorescence (excitation at 282 nm and emission at 331 nm) was measuredat different time points (0–360 min) (broken lines represent control con-ditions without Cu2+ addition). Data are expressed as percentage of theeae

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mission intensity measured before Cu2+ addition. Results are representeds mean ± standard error of mean (S.E.M.) from at least three independentxperiments.

ion was almost completely abolished in the presence of 20nd 30 �M of (PhSe)2.

.5. Aortic slices oxidation

Please cite this article in press as: Bem AF, et al., Diphenyl diselenideoxidation in vitro, Atherosclerosis (2008), doi:10.1016/j.atherosclerosis.

The effects of (PhSe)2 on Cu2+-induced lipid peroxidationn rat aortic slices are depicted in Fig. 5. One-way ANOVAhowed that 10 �M Cu2+ increased (p < 0.01) lipid peroxida-

iilt

rotein and presented as mean ± standard error of mean (S.E.M.) of 3–4ndependent experiments. Controls are represented by an open bar. *p < 0.05;*p < 0.01 (One-way ANOVA followed by the Duncan’s multiple range test).

ion in rat aortic slices. Co-incubation of aortic rats slices withPhSe)2 for 2 h was associated with a reduction in lipid per-xidation induced by copper ions (F(2,13) = 8,67; p < 0.01).PhSe)2 was effective in decreasing Cu2+-induced TBARSormation in a concentration-dependent manner (Fig. 5;= −0.740, p < 0.01).

. Discussion

Oxidative damage to lipoproteins, in particular LDL, isnown to play a role in a number of diseases associated withgeing and is in agreement with the oxidative stress theoryf ageing [30]. Oxidized LDL is involved in the develop-ent of atherosclerosis through the formation of foam cells

31] and autoantibodies against oxidized LDL [32]. SinceDL oxidative damage is involved in the development oftherosclerosis, several recent studies have sought for theotential beneficial effects of antioxidant molecules againstDL oxidation [5,33]. Of particular importance, epidemi-logical studies have pointed to red wine polyphenols asromising molecules that could prevent the development ofeveral coronary syndromes by inhibiting the atherogenicrocess [34].

There is considerable evidence suggesting that an ele-ated plasma copper concentration is associated with existingtherosclerosis and represents a factor of risk for disease inhe future [35]. Catalytically active copper ion has been found

, a simple glutathione peroxidase mimetic, inhibits human LDL2008.02.030

n gruel taken from human advanced atherosclerotic lesions,ndicating that the interior of human advanced atheroscleroticesions is a highly pro-oxidant environment. Consequently,he use of copper ions to promote peroxidation of LDL under

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n vitro conditions can be considered a valid model for study-ng events occurring in atherosclerotic arterial wall [36]. Inddition, AAPH-generated hydroxyl radical has been alsoeported as a good alternative for mimicking oxidative insultshat occur toward lipid and protein moieties of LDL duringhe atherogenic process [37].

Even though several studies have reported the benefi-ial effects of organoseleno compounds against pathologicalonditions associated to oxidative stress (inflammation, gas-ric mucosal damage, neurotoxicity, and hepatotoxicity [11]),here are no reports regarding beneficial effects of (PhSe)2gainst human LDL oxidative damage. In fact, it appearshat ebselen is the only organoseleno compound that haseceived considerable interest during the last decades [14,15].n the present study, the choice for studding (PhSe)2 as aotential beneficial molecule against human LDL oxidationas based on previous studies from our group, which has

hown that (PhSe)2 displays a higher capability of ebselenn detoxifying peroxides at expenses of sulfhydryl molecules21].

Here, we observed that (PhSe)2, an organoseleno com-ound with glutathione peroxidase-like activity, displayedeneficial effects against oxidation induced by copper ionsr AAPH, a hydroxyl radical generator, on human serum andsolated LDL and rat aortic slices. (PhSe)2 increased the oxi-ation lag phase and decreased the oxidation rate in isolateduman LDL and these phenomena were evidenced by usingifferent methodological approaches, including the TBARSeasurement, CDs generation and incorporation of PnA into

he lipoproteins.On the other hand, (PhSe)2 showed an antioxidant effect

n Cu2+-induced oxidation of human serum and this effectas in a concentration-dependent manner. Moreover, this

ffect showed a temporal correlation with the sulfhydrylroups consumption: the increase of TBARS levels startedust when stopped the decrease of SH levels (120 min).his phenomenon suggests the need for available reducedulfhydryl groups for the maintenance of the beneficial effectf (PhSe)2 against Cu2+-induced lipid peroxidation, rein-orcing the idea of the involvement of thiol-peroxidase-likectivity on the antioxidant effects of (PhSe)2.

This idea is reinforced by the observed beneficial effects ofPhSe)2 against the oxidation of endogenous and exogenousincorporated PnA) LDL lipids. Inline with this, spec-roscopic studies showed the direct chemical interactionetween (PhSe)2 and GSH, resulting in the formation of ahemical intermediary whose stability is affected by the pres-nce of hydrogen peroxide. Furthermore, we demonstratedconcentration-dependent antioxidant effect of (PhSe)2 onu2+-oxidized rat aortic slices, indicating its antiatherogenicotential. Therefore, (PhSe)2 behaves as a potent antioxi-ant in different models intimately related to the atherogenic

Please cite this article in press as: Bem AF, et al., Diphenyl diselenideoxidation in vitro, Atherosclerosis (2008), doi:10.1016/j.atherosclerosis.

rocess.An interesting effect of this study was that (PhSe)2 dis-

layed significant beneficial effects in the different phasesf Cu2+-induced LDL oxidation. In fact, when added to the

PRESSis xxx (2008) xxx–xxx

eaction medium at time zero (Fig. 3A), (PhSe)2 signifi-antly prevented LDL oxidation. Moreover, when added tohe reaction medium at 45 or 90 min (partially oxidized LDL),PhSe)2 was capable of stopping CDs formation. Mecha-istically, these results are of interest because they indicatehat the thiol-peroxidase activity of (PhSe)2 is enough impor-ant to prevent the generation of secondary products of lipideroxidation, such as CDs.

Another significant (and maybe the most important) resultrom our study was the capability of (PhSe)2 to prevent Cu2+-nduced loss of Trp fluorescence in human LDL. In thisegard, it has been reported that the fluorescence spectrum ofative LDL displays a single band centered at approximately32 nm, which is assigned to the Trp residues in apo B andoss of Trp fluorescence is a marker for oxidations at the pro-ein core of LDL [29]. The protective effect of (PhSe)2 againstu2+-induced loss of Trp fluorescence indicates that, besides

t’s beneficial effects against oxidation of lipid moieties ofDL, this chalcogen also prevents the oxidation of proteinoieties of human LDL, pointing to an additional mecha-

ism that could contribute to the inhibition of the atherogenicrocess.

The results of the present study are the first to show that (i)PhSe)2 inhibits lipid peroxidation in human isolated LDL initro, (ii) this phenomenon is related to its thiol-peroxidasectivity, and (iii) this chalcogen also prevents the oxidationf protein moieties of human LDL. Taken together, such dataender (PhSe)2 a promising molecule for pharmacologicaltudies with respect to the atherogenic process.

cknowledgements

The authors are grateful to Dr. Gilson Zeni to provideiphenyl disselenide for assays. The financial support byFSM (FIPE), FAPERGS, CAPES and CNPq are gratefully

cknowledged. M.F., J.B.T.R. and C.W.N. are the recipientsf CNPq fellowships.

eferences

[1] Matsuura E, Kobayashi K, Tabuchi M, Lopez LR. Oxidativemodification of low-density lipoprotein and immune regulation ofatherosclerosis. Prog Lipid Res 2006;45:466–86.

[2] Witzum JL. The oxidation hypothesis of atherosclerosis. Lancet1994;344:793–5.

[3] Halliwell B. Oxidation of low-density lipoproteins: questions of ini-tiation, propagation, and the effect of antioxidants. Am J Clin Nutr1995;61(Suppl. 3):670S–7S.

[4] Esterbauer H, Gebicki J, Puhl H, Jurgens G. The role of lipid peroxi-dation and antioxidants in oxidative modification of LDL. Free RadicBiol Med 1992;13:341–90.

[5] Noguchi N, Niki E. Phenolic antioxidants: a rationale for design and

, a simple glutathione peroxidase mimetic, inhibits human LDL2008.02.030

evaluation of novel antioxidant drug for atherosclerosis. Free RadicBiol Med 2000;28:1538–46.

[6] Hu ML, Tappel AL. Selenium as a sulfhydryl redox catalyst andsurvey of potential selenium-dependent enzymes. Inorg Biochem1987;30:239–48.

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[7] Suadicani P, Hein HO, Gyntelberg F. Serum selenium concentrationand risk of ischaemic heart disease in a prospective cohort study of3000 males. Atherosclerosis 1992;96:33–42.

[8] Thomas JP, Geiger PG, Girotti AW. Lethal damage to endothelial cellsby oxidized low density lipoprotein: role of selenoperoxidases in cyto-protection against lipid hydroperoxide- and iron-mediated reactions. JLipid Res 1993;34:479–90.

[9] Andersson CM, Hallberg A, Linden M, et al. Antioxidant activityof some diarylselenides in biological systems. Free Radic Biol Med1994;16:17–28.

10] Wilson SR, Zucker PA, Huang RRC, Spector A. Development of syn-thetic compounds with glutathione peroxidase activity. J Am Chem Soc1989;111:5936–9.

11] Nogueira CW, Zeni G, Rocha JB. Organoselenium and organ-otellurium compounds: toxicology and pharmacology. Chem Rev2004;104:6255–85.

12] Muller A, Cadenas E, Graf P, Sies H. A novel biologically activeseleno-organic compound. I. Glutathione peroxidase-like activity invitro and antioxidant capacity of PZ 51 (Ebselen). Biochem Pharmacol1984;33:3235–9.

13] Daiber A, Zou MH, Bachschmid M, Ullrich V. Ebselen as a per-oxynitrite scavenger in vitro and ex vivo. Biochem Pharmacol2000;59:153–60.

14] Saito I, Asano T, Sano K, et al. Neuroprotective effect of an antioxidant,ebselen, in patients with delayed neurological deficits after aneurysmalsubarachnoid hemorrhage. Neurosurgery 1998;42:269–77.

15] Yamaguchi T, Sano K, Takakura K, et al. Ebselen in acute ischemicstroke: a placebo-controlled, double-blind clinical trial. Ebselen StudyGroup. Stroke 1998;29:12–7.

16] Meotti FC, Stangherlin EC, Zeni G, Nogueira CW, Rocha JB. Protectiverole of aryl and alkyl diselenides on lipid peroxidation. Environ Res2004;94:276–82.

17] Nogueira CW, Meotti FC, Curte E, et al. Investigations into the poten-tial neurotoxicity induced by diselenides in mice and rats. Toxicology2003;183:29–37.

18] Bem AF, Portella RL, Perottoni J, et al. Changes in biochemical param-eters in rabbits blood after oral exposure to diphenyl diselenide for longperiods. Chem Biol Interact 2006;162:1–10.

19] Bem AF, Portella RL, Farina M, et al. Low toxicity of diphenyl dis-elenide in rabbits: a long-term study. Basic Clin Pharmacol Toxicol2007;101:47–55.

Please cite this article in press as: Bem AF, et al., Diphenyl diselenideoxidation in vitro, Atherosclerosis (2008), doi:10.1016/j.atherosclerosis.

20] Barbosa NB, Rocha JB, Wondracek DC, et al. Diphenyl diselenidereduces temporarily hyperglycemia: possible relationship with oxida-tive stress. Chem Biol Interact 2006;163:230–8.

21] Posser T, Moretto MB, Dafre AL, et al. Antioxidant effect of diphenyldiselenide against sodium nitroprusside (SNP) induced lipid perox-

[

PRESSis xxx (2008) xxx–xxx 9

idation in human platelets and erythrocyte membranes: an in vitroevaluation. Chem Biol Interact 2006;164:126–35.

22] Paulmier C. Selenium reagents and intermediates in organic synthesis.New York: Pergamon Books; 1986. p. 463.

23] Ohkawa H, Ohishi H, Yagi K. Assay for lipid peroxide in animal tissuesby thiobarbituric acid reaction. Anal Biochem 1979;95:351–8.

24] Ellman GL. Tissue sulphydryl groups. Arch Biochem Biophys1959;82:70.

25] Silva EL, Tsushida T, Terao J. Inhibition of mammalian 15-lipoxygenase-dependent LDL lipid peroxidation by quercetin andquercetin-monoglucosides. Arch Biochem Biophys 1998;349:313–20.

26] Lowry OH, Rosebrough NJ, Farr AL, Randall RJ. Protein measurementwith the Folin phenol reagent. J Biol Chem 1951;193:265–75.

27] Ziouzenkova O, Sevanian A, Abuja PM, Ramos P, Esterbauer H. Coppercan promote oxidation of LDL by markedly different mechanisms. FreeRadic Biol Med 1998;24:607–23.

28] Laranjinha JAN, Almeida LM, Madeira VMC. Lipid peroxidation andits inhibition in low density lipoproteins: quenching of cis-parinaricacidfluorescence. Arch Biochem Biophys 1992;297:147–54.

29] Reyftmann JP, Santus R, Maziere JC, et al. Sensitivity of tryptophaneand related compounds to oxidation induced by lipid autoperoxida-tion. Application to human low- and high-density lipoproteins. BiochimBiophys Acta 1990;1042:159–67.

30] Stocker R, Keaney Jr JF. Role of oxidative modifications in atheroscle-rosis. Physiol Rev 2004;84:1381–478.

31] Parthasarathy S, Quinn MT, Steinberg D. Is oxidized low den-sity lipoprotein involved in the recruitment and retention ofmonocyte/macrophages in the artery wall during the initiation ofatherosclerosis? Basic Life Sci 1988;49:375–80.

32] Faviou E, Vourli G, Nounopoulos C, Zachari A, Dionyssiou-AsteriouA. Circulating oxidized low density lipoprotein, autoantibodies againstthem and homocysteine serum levels in diagnosis and estimationof severity of coronary artery disease. Free Radic Res 2005;39:419–29.

33] Kaliora AC, Dedoussis GVZ, Schmidt H. Dietary antioxidants in pre-venting atherogenesis. Atherosclerosis 2006;187:1–17.

34] Ursini F, Sevanian A. Wine polyphenols and optimal nutrition. Ann NYAcad Sci 2002;957:200–9.

35] Ferns GAA, Lamb DJ, Taylor A. The possible role of copper ions inatherogenesis: the Blue Janus. Atherosclerosis 1997;133:139–52.

36] Smith C, Mitchinson MJ, Aruoma OI, et al. Stimulation of lipid per-

, a simple glutathione peroxidase mimetic, inhibits human LDL2008.02.030

oxidation and hydroxyl-radical generation by the contents of humanatherosclerotic lesions. Biochem J 1992;286:901–5.

37] Atkin MA, Gasper A, Ullegaddi R, Powers HJ. Oxidative susceptibilityof unfractionated serum or plasma: response to antioxidants in vitro andto antioxidant supplementation. Clin Chem 2005;51:2138–44.